This invention relates, in part, to newly identified polynucleotides and polypeptides; variants and derivatives of these polynucleotides and polypeptides; processes for making these polynucleotides and these polypeptides, and their variants and derivatives; agonists and antagonists of the polypeptides; and uses of these polynucleotides, polypeptides, variants, derivatives, agonists and antagonists. In particular, in these and in other regards, the invention relates to polynucleotides and polypeptides of staphylococcal FabI enoyl-ACP reductase hereinafter referred to as xe2x80x9cFABIxe2x80x9d.
Although the overall pathway of saturated fatty acid biosynthesis is similar in all organisms, the fatty acid synthase (FAS) systems vary considerably with respect to their structural organization. Thus in Type I FAS systems, found in vertebrates and yeasts, the necessary enzymes required for fatty acid synthesis are present on one or two polypeptide chains respectively. In contrast, in Type II systems found in most bacteria and plants, each step in the pathway is catalysed by a separate mono-functional enzyme. It would therefore appear that significant selectivity of inhibition of the bacterial and mammalian enzymes is possible.
FabI (previously designated EnvM) functions as an enoyl-acyl carrier protein (ACP) reductase (Bergler, et al, (1994), J. Biol. Chem. 269, 5493-5496) in the final step of the four reactions involved in each cycle of bacterial fatty acid biosynthesis.
The first step is catalysed by xcex2-ketoacyl-ACP synthase, which condenses malonyl-ACP with acetyl-CoA (FabH, synthase III). In subsequent rounds malonyl-ACP is condensed with the growing-chain acyl-ACP (FabB and FabF, synthases I and II respectively).
The second step in the elongation cycle is ketoester reduction by NADPH-dependent xcex2-ketoacyl-ACP reductase (FabG). Subsequent dehydration by xcex2-hydroxyacyl-ACP dehydrase (either FabA or FabZ) leads to trans-2-enoyl-ACP which is in turn converted to acyl-ACP by NADH-dependent enoyl-ACP reductase (FabI). Further rounds of this cycle, adding two carbon atoms per cycle, eventually lead to palmitoyl-ACP (16C) where upon the cycle is stopped largely due to feedback inhibition of FabI by palmitoyl-ACP (Heath, et al, (1996), J. Biol. Chem. 271, 1833-1836). FabI is therefore a major biosynthetic enzyme which is also a key regulatory point in the overall synthetic pathway.
Early data suggested that there were two enoyl-ACP reductases in E. coli, one NADPH dependent and the other NADH dependent. However, more recent work has found no evidence for the NADPH dependent enzyme and FabI is the only enoyl ACP reductase identified in E. coli. (Heath, et al, (1995), J. Biol. Chem. 270, 26538-26542; Bergler, et al, (1994), J. Biol. Chem. 269, 5493-5496).
It has been shown that diazaborine antibiotics inhibit fatty acid, phospholipid and lipopolysaccharide (LPS) biosynthesis and it has also been shown that the antibacterial target of these compounds is FabI. For example derivative 2b18 from Grassberger, et al (1984) J. Med Chem 27 947-953 has been shown to be a non-competitive inhibitor of FabI having a Ki=0.2 mM (Bergler, et al, (1994), J. Biol. Chem. 269, 5493-5496). The antibacterial activity of diazaborine derivatives against Gram-negatives and Gram positive organisms is well documented (Grassberger et al., J Med Chem. 1984 27, 947-953; Gronowitz et al., Acta Pharm Suecica, 1971 8 377; Wersch et al U.S. Pat. No. 2,533,918; Lam et al., J. Antimirob. Chemother. 1987 20 37-45).
Conditionally lethal FabI mutants have been constructed in E. coli and the FabI gene from Salmonella typhimurium complements this mutation. In addition, plasmids containing the FabI gene from diazaborine resistant S. typhimurium conferred diazaborine resistance in E. coli (Turnowsky, et al, (1989), J. Bacteriol., 171, 6555-6565) confirming FabI as the antibacterial target of diazaborines.
Inhibition of FabI either by diazaborine or by raising the temperature in an FabI temperature sensitive mutant to non-permissive conditions is lethal, thus demonstrating that FabI is essential to the survival of the organism (Bergler, et al, (1994), J. Biol. Chem. 269, 5493-5496). Laboratory generated point mutations in the FabI gene lead to diazaborine resistant E. coli. 
FabI is conserved in Gram negative organisms with 98% identity between E. coli and S. typhimurium FabI (Bergler, et al, (1992), J. Gen. Microbiol., 138, 2093-2100) and 75% identity between these proteins and H. influenzae FabI. Staphylococcus aureus FABI of the invention shows 54% similarity to the mycobacterial protein, InhA, which is highly conserved throughout mycobacteria including M. tuberculosis. E. coli FabI was found to be 34% identical, 57% similar to Brassica napus (rape seed) enoyl-ACP reductase and S. aureus FABI of the present invention was also 34% identical, 57% similar. Moreover, FABI of the present invention was found to be 44% identical, 64% similar over 252 amino acids to E. coli FabI. FABI of the present invention is only 27% identical, 48% similar to a mammalian 2,4-dienoyl-coenzyme A reductase. This mammalian homolog differs from FABI in that it is involved in the xcex2-oxidation of polyunsaturated enoyl-CoAs and utilizes NADPH as cofactor rather than NADH. Therefore, there is significant potential for selective inhibition of FABI. Since there are no marketed antibiotics targeted against fatty acid biosynthesis it is likely that inhibitors of FABI will not be susceptible to current antibiotic resistance mechanisms. Moreover, this is a potentially broad spectrum target.
There is an unmet need for developing new classes of antibiotic compounds. Clearly, there is also a need for factors, such as FABI that may be used to screen compounds for antibiotic activity, such as a simple high through-put assay for screening inhibitors of FAS. Such factors may also be used to determine their roles in pathogenesis of infection, dysfunction and disease. Identification and characterization of such factors, which can play a role in preventing, ameliorating or correcting infections, dysfunctions or diseases are critical steps in making important discoveries to improve human health.
Toward these ends, and others, it is an object of the present invention to provide polypeptides, inter alia, that have been identified as novel FABI by homology between the amino acid sequence set out in FIG. 1 [SEQ ID NO:2] and known amino acid sequences of other proteins such as E. coli FabI enoyl-ACP reductase and those afforementioned.
It is a further object of the invention, moreover, to provide polynucleotides that encode FABI, particularly polynucleotides that encode the polypeptide herein designated FABI.
In a particularly preferred embodiment of this aspect of the invention the polynucleotide comprises the region encoding FABI in the sequence set out in FIG. 1 [SEQ ID NO:2].
In another particularly preferred embodiment of the present invention there is a novel FABI protein from S. aureus WCUH 29 comprising the amino acid sequence of SEQ ID NO:2, or a fragment, analogue or derivative thereof.
In accordance with this aspect of the invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Staphylococcus aureus WCUH 29 strain contained in NCIMB Deposit No. 40771.
In accordance with this aspect of the invention there are provided isolated nucleic acid molecules encoding FABI, particularly staphylococcal FABI, including mRNAs, cDNAs, genomic DNAs and, in further embodiments of this aspect of the invention, biologically. diagnostically, clinically or therapeutically useful variants, analogs or derivatives thereof, or fragments thereof, including fragments of the variants, analogs and derivatives.
Among the particularly preferred embodiments of this aspect of the invention are naturally occurring allelic variants of FABI.
In accordance with this aspect of the invention there are provided novel polypeptides of staphylococcal origin referred to herein as FABI as well as biologically, diagnostically or therapeutically useful fragments, variants and derivatives thereof, variants and derivatives of the fragments, and analogs of the foregoing.
It also is an object of the invention to provide FABI polypeptides, particularly FABI polypeptides, that may be employed for therapeutic purposes, for example, to treat disease, including treatment by conferring host immunity against infections, such as staphlococcal infections including, but not limited to infections of upper respiratory tract (e.g. otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g. empyema, lung abscess), cardiac (e.g. infective endocarditis), gastrointestinal (e.g. secretory diarrhoea, splenic abscess, retroperitoneal abscess), CNS (e.g. cerebral abscess), eye (e.g. blepharitis, conjunctivitis, keratitis, endophalmitis, preseptal and orbital cellulitis, darcryocystitis), kidney and urinary tract (e.g. epidymitis, intrarenal and perinephric abscess, toxic shock syndrome), skin (e.g. impetigo, folliculitis, cutaneous abscesses, cellulitis, wound infection, bacterial myositis), and bone and joint (e g. septic arthritis, osteomyelitis).
In accordance with yet a further aspect of the present invention, there is provided the use of a polypeptide of the invention for therapeutic or prophylactic purposes, for example, as an antibacterial agent or a vaccine.
In accordance with another aspect of the present invention, there is provided the use of a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization.
Among the particularly preferred embodiments of this aspect of the invention are variants of FABI polypeptide encoded by naturally occurring alleles of the FABI gene.
It is another object of the invention to provide a process for producing the aforementioned polypeptides, polypeptide fragments, variants and derivatives, fragments of the variants and derivatives, and analogs of the foregoing.
In a preferred embodiment of this aspect of the invention there are provided methods for producing the aforementioned FABI polypeptides comprising culturing host cells having expressibly incorporated therein an exogenously-derived FABI-encoding polynucleotide under conditions for expression of FABI in the host and then recovering the expressed polypeptide.
In accordance with another object the invention there are provided products, compositions, processes and methods that utilize the aforementioned polypeptides and polynucleotides for research, biological, clinical and therapeutic purposes, inter alia.
In accordance with yet another aspect of the present invention, there are provided inhibitors to such polypeptides, useful as antibacterial agents. In particular, there are provided antibodies against such polypeptides.
In accordance with certain preferred embodiments of this aspect of the invention, there are provided products, compositions and methods, inter alia, for, among other things: assessing FABI expression in cells by detecting FABI polypeptides or FABI-encoding mRNA; to treat bacterial infections in vitro, ex vivo or in vivo by exposing cells to FABI polypeptides or polynucleotides as disclosed herein; assaying genetic variation and aberrations, such as defects, in FABI genes; and administering a FABI polypeptide or polynucleotide to an organism to raise an immunological response against bacteria, such as, for example a staphylococcus.
In accordance with certain preferred embodiments of this and other aspects of the invention there are probes that hybridize to FABI sequences.
In certain additional preferred embodiments of this aspect of the invention there are provided antibodies against FABI polypeptides. In certain particularly preferred embodiments in this regard, the antibodies are highly selective for FABI.
In accordance with another aspect of the present invention, there are provided FABI agonists. Among preferred agonists awe molecules that mimic FABI, that bind to FABI-binding molecules or binding molecules, and that elicit or augment FABI-induced responses. Also among preferred agonists are molecules that interact with FABI, or with other modulators of FABI activities, and thereby potentiate or augment an effect of FABI or more than one effect of FABI and are bacteriostatic or bacteriocidal.
In accordance with yet ether aspect of the present invention, there are provided FABI antagonists. Among preferred antagonists are those which mimic FABI so as to bind to FABI-binding molecules but not elicit a FABI-induced response or more than one FABI-induced response. Also among preferred antagonists are molecules that bind to or interact with FABI so as to inhibit an effect of FABI or more than one effect of FABI or which prevent expression of FABI. Further particularly preferred antagonists of FABI lower or abolish a FABI enzymatic activity or activities.
In a further aspect of the invention there are provided compositions comprising a FABI polynucleotide or a FABI polypeptide for administration to cells in vitro, to cells ex vivo and to cells in vivo, or to a multicellular organism. In certain particularly preferred embodiments of this aspect of the invention, the compositions comprise a FABI polynucleotide for expression of a FABI polypeptide in a host organism to raise an immunological response, preferably to raise immunity in such host against bacteria, preferably staphylocci or closely genetically related organisms.
Other objects, features, advantages and aspects of the present invention will become apparent to those of skill from the following description. It should be understood, however, that the following description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following description and from reading the other parts of the present disclosure.